The development of a bioengineered heparin from a non-animal source is in response to a health crisis that took place in early 2008. This crisis involved the introduction of an oversulfated chondroitin sulfate into heparin produced from hogs in China leading to the death of nearly 100 Americans. Recent research in our laboratories suggests that it is now possible to prepare a bioengineered heparin from non-animal sources using fermentation combined with chemoenzymatic methods. The proposed 5-year project is a translational and multi-disciplinary research effort involving the Rensselaer Polytechnic Institute, University of North Carolina and Albany College of Pharmacy, aimed at producing kilogram quantities of non-animal sourced bioengineered heparin. By controlling the process steps this bioengineered heparin will be prepared with a structure identical to the pharmaceutical heparin prepared from animals. Both chemical and bioequivalence studies will provide the necessary pre-clinical data required to carry bioengineered heparin forward as a generic heparin. The results of this 5-year translational bioengineering research project will be the synthesis of 1 kilogram of non-animal sourced heparin, which serves as a well defined deliverable that is chemically and biologically equivalent to USP heparin. A second well-defined deliverable will be an optimized and cost effective process that can be used ultimately to generate bioengineered, non-animal heparin at scales sufficient to satisfy the therapeutic needs in the US. This material and the accompanying process will be made available to both large and small business partners interested in moving this bioengineered heparin into human clinical trails as a novel and safer replacement for animal sourced heparin. We hypothesize that application of recombinantly-expressed biosynthetic enzymes in a well controlled process can afford a bioengineered heparin that is the generic equivalent of USP heparin. Furthermore, we envision that this bioengineered heparin will be safer for patients and can be prepared at costs competitive to heparin obtained from animal tissues. There are four specific aims of this proposal. 1. Optimize the production of bioengineered heparin; 2. Confirm chemical equivalence of bioengineered heparin with USP heparin; 3. Confirm bioequivalence of bioengineered heparin with USP heparin; and 4. Scale-up and produce a kilogram of bioengineered heparin while maintaining chemical and bioequivalence.